Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples.

BACKGROUND: Sequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively. RESULTS: With both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing. CONCLUSIONS: The results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Changes in liver metabolic pathways demonstrate efficacy of the combined dietary and microbial therapeutic intervention in MASLD mouse model.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease globally, yet no therapies are approved. The effects of Escherichia coli Nissle 1917 expressing aldafermin, an engineered analog of the intestinal hormone FGF19, in combination with dietary change were investigated as a potential treatment for MASLD. METHODS: MASLD was induced in C57BL/6J male mice by American lifestyle-induced obesity syndrome diet and then switched to a standard chow diet for seven weeks. In addition to the dietary change, the intervention group received genetically engineered E. coli Nissle expressing aldafermin, while control groups received either E. coli Nissle vehicle or no treatment. MASLD-related plasma biomarkers were measured using an automated clinical chemistry analyzer. The liver steatosis was assessed by histology and bioimaging analysis using Fiji (ImageJ) software. The effects of the intervention in the liver were also evaluated by RNA sequencing and liquid-chromatography-based non-targeted metabolomics analysis. Pathway enrichment studies were conducted by integrating the differentially expressed genes from the transcriptomics findings with the metabolites from the metabolomics results using Ingenuity pathway analysis. RESULTS: After the intervention, E. coli Nissle expressing aldafermin along with dietary changes reduced body weight, liver steatosis, plasma aspartate aminotransferase, and plasma cholesterol levels compared to the two control groups. The integration of transcriptomics with non-targeted metabolomics analysis revealed the downregulation of amino acid metabolism and related receptor signaling pathways potentially implicated in the reduction of hepatic steatosis and insulin resistance. Moreover, the downregulation of pathways linked to lipid metabolism and changes in amino acid-related pathways suggested an overall reduction of oxidative stress in the liver. CONCLUSIONS: These data support the potential for using engineered microbial therapeutics in combination with dietary changes for managing MASLD.

Genome sequences of BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c.

BACKGROUND: Bacillus Calmette-Guérin (BCG) remains the only vaccine to prevent tuberculosis (TB) during childhood, with relatively low to no efficacy against pulmonary TB in adolescents and adults. BCG consists of close to 15 different substrains, where genetic variations among them might contribute to the variable protective efficacy afforded against pulmonary TB. We have shown that the vaccine candidate, BCGΔBCG1419c, which is based on BCG Pasteur, improved protection against chronic TB in murine models, as well as against pulmonary and extrapulmonary TB in guinea pigs. Here, to confirm deletion of the BCG1419c gene and to detect possible genetic variations occurring as a consequence of the spontaneous mutations that may arise during in vitro culture of mycobacteria, the genomes of BCG Pasteur ATCC 35734 and its isogenic derivative, BCGΔBCG1419c, were sequenced and subjected to a comparative analysis between them and against BCG Pasteur 1173P2. RESULTS: The complete catalog of variants in genes relative to the reference genome BCG Pasteur 1173P2 (GenBank NC008769) showed that the parental strain BCG Pasteur ATCC 35734, from which the mutant BCGΔBCG1419c originated, showed five synonymous mutations, three missense mutations, and five codon insertions, whereas the BCGΔBCG1419c mutant reported the same changes. When BCG Pasteur ATCC 35734 and BCGΔBCG1419c were compared, we confirmed that the latter was devoid of the BCG1419c gene, with only one unanticipated SNP at position 2, 828, 791  which we consider has no role in vaccine properties reported thus far. CONCLUSION: We provide evidence that the mutagenesis performed to remove BCG1419c from BCG Pasteur ATCC 35734 solely deleted this gene, and that compared with the reference strain BCG Pasteur 1173P2, few changes were present confirming that they are BCG Pasteur strains, and that changes in immunogenicity or efficacy observed thus far in BCGΔBCG1419c are most likely derived solely from the elimination of the BCG1419c gene.

Complete Genome Sequences of Two Lytic Phages of Salmonella enterica Isolated from Wastewater in Ecuador.

Salmonella enterica is one of the most common causes of foodborne diseases. Bacteriophages provide an option to reduce the presence of Salmonella. Here, we describe the isolation of two lytic Salmonella bacteriophages. The complete genomes were annotated and show similarity to that of the lytic phage NBSal001, in the Drexlerviridae family.

Influence of perinatal and childhood exposure to tobacco and mercury in children's gut microbiota.

Background: Early life determinants of the development of gut microbiome composition in infants have been widely investigated; however, if early life pollutant exposures, such as tobacco or mercury, have a persistent influence on the gut microbial community, its stabilization at later childhood remains largely unknown. Objective: In this exposome-wide study, we aimed at identifying the contribution of exposure to tobacco and mercury from the prenatal period to childhood, to individual differences in the fecal microbiome composition of 7-year-old children, considering co-exposure to a width of established lifestyle and clinical determinants. Methods: Gut microbiome was studied by 16S rRNA amplicon sequencing in 151 children at the genus level. Exposure to tobacco was quantified during pregnancy through questionnaire (active tobacco consumption, second-hand smoking -SHS) and biomonitoring (urinary cotinine) at 4 years (urinary cotinine, SHS) and 7 years (SHS). Exposure to mercury was quantified during pregnancy (cord blood) and at 4 years (hair). Forty nine other potential environmental determinants (12 at pregnancy/birth/infancy, 15 at 4 years and 22 at 7 years, such as diet, demographics, quality of living/social environment, and clinical records) were registered. We used multiple models to determine microbiome associations with pollutants including multi-determinant multivariate analysis of variance and linear correlations (wUnifrac, Bray-Curtis and Aitchison ß-diversity distances), single-pollutant permutational multivariate analysis of variance adjusting for co-variates (Aitchison), and multivariable association model with single taxa (MaAsLin2; genus). Sensitivity analysis was performed including genetic data in a subset of 107 children. Results: Active smoking in pregnancy was systematically associated with microbiome composition and ß-diversity (R2 2-4%, p < 0.05, Aitchison), independently of other co-determinants. However, in the adjusted single pollutant models (PERMANOVA), we did not find any significant association. An increased relative abundance of Dorea and decreased relative abundance of Akkermansia were associated with smoking during pregnancy (q < 0.05). Discussion: Our findings suggest a long-term sustainable effect of prenatal tobacco exposure on the children's gut microbiota. This effect was not found for mercury exposure or tobacco exposure during childhood. Assessing the role of these exposures on the children's microbiota, considering multiple environmental factors, should be further investigated.

Rapid Oxford Nanopore Technologies MinION Sequencing Workflow for Campylobacter jejuni Identification in Broilers on Site­A Proof-of-Concept Study

Campylobacter is recognised as one of the most important foodborne bacteria, with a worldwide health and socioeconomic impact. This bacterium is one of the most important zoonotic players in poultry, where efficient and fast detection methods are required. Current official culture methods for Campylobacter enumeration in poultry usually include >44 h of culture and >72 h for identification, thus requiring at least five working shifts (ISO/TS 10272-2:2017). Here, we have assembled a portable sequencing kit composed of the Bento Lab and the MinION and developed a workflow for on-site farm use that is able to detect and report the presence of Campylobacter from caecal samples in less than five hours from sampling time, as well as the relationship of Campylobacter with other caecal microbes. Beyond that, our workflow may offer a cost-effective and practical method of microbiologically monitoring poultry at the farm. These results would demonstrate the possibility of carrying out rapid on-site screening to monitor the health status of the poultry farm/flock during the production chain.

Evolutionary and Phenotypic Characterization of Two Spike Mutations in European Lineage 20E of SARS-CoV-2.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants.

The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

Analysis of the Gut Microbiota: An Emerging Source of Biomarkers for Immune Checkpoint Blockade Therapy in Non-Small Cell Lung Cancer.

BACKGROUND: The human gut harbors around 1013-1014 microorganisms, collectively referred to as gut microbiota. Recent studies have found that the gut microbiota may have an impact on the interaction between immune regulation and anti-cancer immunotherapies. METHODS: In order to characterize the diversity and composition of commensal microbiota and its relationship with response to immune checkpoint blockade (ICB), 16S ribosomal DNA (rDNA) sequencing was performed on 69 stool samples from advanced non-small cell lung cancer (NSCLC) patients prior to treatment with ICB. RESULTS: The use of antibiotics and ICB-related skin toxicity were significantly associated with reduced gut microbiota diversity. However, antibiotics (ATB) usage was not related to low ICB efficacy. Phascolarctobacterium was enriched in patients with clinical benefit and correlated with prolonged progression-free survival, whereas Dialister was more represented in patients with progressive disease, and its higher relative abundance was associated with reduced progression-free survival and overall survival, with independent prognostic value in multivariate analysis. CONCLUSIONS: Our results corroborate the relation between the baseline gut microbiota composition and ICB clinical outcomes in advanced NSCLC patients, and provide novel potential predictive and prognostic biomarkers for immunotherapy in NSCLC.

Effect of Mastiha supplementation on NAFLD: The MAST4HEALTH Randomised, Controlled Trial.

SCOPE: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease with poor therapeutic strategies. Mastiha possesses antioxidant/anti-inflammatory and lipid-lowering properties. The authors investigate the effectiveness of Mastiha as a nonpharmacological intervention in NAFLD. METHODS AND RESULTS: Ninety-eight patients with NAFLD in three countries (Greece, Italy, Serbia) are randomly allocated to either Mastiha or Placebo for 6 months, as part of a multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical trial. The authors assess NAFLD severity via magnetic resonance imaging (MRI) scanning and LiverMultiScan technique and evaluate the effectiveness of Mastiha through medical, anthropometric, biochemical, metabolomic, and microbiota assessment. Mastiha is not superior to Placebo on changes in iron-corrected T1 (cT1) and Liver Inflammation Fibrosis score (LIF) in entire patient population; however, after BMI stratification (BMI ≤ 35 kg m-2 and BMI > 35 kg m-2 ), severely obese patients show an improvement in cT1 and LIF in Mastiha versus Placebo. Mastiha increases dissimilarity of gut microbiota, as shown by the Bray-Curtis index, downregulates Flavonifractor, a known inflammatory taxon and decreases Lysophosphatidylcholines-(LysoPC) 18:1, Lysophosphatidylethanolamines-(LysoPE) 18:1, and cholic acid compared to Placebo. CONCLUSION: Mastiha supplementation improves microbiota dysbiosis and lipid metabolite levels in patients with NAFLD, although it reduces parameters of liver inflammation/fibrosis only in severely obese patients.

Profiling the Bladder Microbiota in Patients With Bladder Cancer.

Evidence suggests that microbiota may contribute to the pathogenesis of several diseases, including cancer. In the case of bladder cancer, preliminary studies have found alterations in the urinary microbiota of patients with urothelial carcinoma compared with healthy individuals. Conversely, the urinary microbiota differ between men and women, and it has been hypothesized that these differences are associated with the lower incidence of bladder cancers in women. The objective of this study was to characterize the bladder microbiota in paired samples of tumor and non-tumor mucosa of patients with malignant bladder neoplasia using next-generation sequencing. In addition, we aimed to study potential differences in microbial composition in tumor samples according to clinical and pathological variables, and to determine possible microbial profiles. We found significant differences in microbial richness at the genus level, with a higher richness observed in the non-tumor compared with the tumor mucosa. It was also shown that Actinobacteria were significantly more enriched in the non-tumor compared with the tumor mucosa (P = 0.014). In the multivariate analysis, we found significant differences in microbial composition according to tumor grade (P = 0.03 and 0.04 at the phylum and genus levels, respectively). Moreover, we detected a higher microbial richness in non-tumor vs. tumor tissues which agrees with the global assumption that microbial richness is an indicator of health. The greater abundance of members of the Actinobacteria phylum in the non-neoplastic bladder mucosa samples supports the hypothesis that a higher abundance of Actinomycetes is associated with a lower rate of bladder cancer in women and suggests a protective role for these microbiota. We detected a microbial profile that was enriched for Enterococcus in low-grade tumors. Finally, we identified the presence of two clusters in the microbial composition of the tumor mucosa samples, significantly enriched for the genera Barnesiella, Parabacteroides, Prevotella, Alistipes, and Lachnospiracea_incertae_sedis (Cluster 1), or Staphylococcus (Cluster 2). Further longitudinal studies are needed to assess the role of the bladder microbiota in carcinogenesis.

Roles of host genetics and sperm microbiota in reproductive success in healthy rabbit.

Although the effects of sperm microbiota and sperm quality have been described previously, recent studies provide evidence that female genital modifications triggered by seminal components could be of significant importance to identify some disturbances associated with fertility. So, sperm microbiota could play a key role in sperm quality, contributing to fertilisation. To understand how sperm microbiota diversity is influenced by the host genetics, the symbiotic bacteria in four inbred lines raised in the same animal facility and their effects on sperm quality and fertility were analysed. Forty healthy rabbits from four selected Spanish commercial lines were used in this research (three based on litter performance, designated A, V and LP, and one selected for daily body weight gain, called R). Significant variations in the seminal concentration, morphology and some motion parameters were found among inbred lines, but sperm motility and viability were similar among inbred lines. After mating, inbred lines selected for litter size had the same fertility rate, significantly higher than inbred line selected for body weight (82 ± 3.3%, 79 ± 3.5% and 89 ± 4.5% versus 61 ± 3.7%, for the A, V and LP vs R lines, respectively, p < 0.05). Bacteria belonging to Proteobacteria, Firmicutes, Fusobacteria and Bacteroidetes were identified in sperm microbiota. At genus level, the bacterial community composition in the sperm microbiota was influenced by host genetics. A total of 35, 16, 34, and 51 genera were accurately detected in the A, V, LP, and R lines, respectively. Moreover, Enhydrobacter, Ferruginibacter, Myroides Paracoccus, Rheinheimera, Tepidiphilus, Tetradesmus obliquus and Thauera genera were present only in the inbred lines selected for litter size. Moreover, the discriminant analysis revealed Lysinibacillus and Flavobacterium genera as potential biomarkers for fertility. Thus, these two genera may play a key role in fertility. Our results demonstrated the existence of a rabbit inbred line-specific variation in bacterial occurrence in sperm microbiota. Moreover, fertility differentials among inbred lines that were not predicted by routine semen analysis could be partly explained by the symbiotic state of the semen microbiota.

Detection of mixed-strain infections by FACS and ultra-low input genome sequencing.

The epidemiological tracking of a bacterial outbreak may be jeopardized by the presence of multiple pathogenic strains in one patient. Nevertheless, this fact is not considered in most of the epidemiological studies and only one colony per patient is sequenced. On the other hand, the routine whole genome sequencing of many isolates from each patient would be costly and unnecessary, because the number of strains in a patient is never known a priori. In addition, the result would be biased by microbial culture conditions. Herein we propose an approach for detecting mixed-strain infection, providing C. difficile infection as an example. The cells of the target pathogenic species are collected from the bacterial suspension by the fluorescence activated cell sorting (FACS) and a shallow genome sequencing is performed. A modified sequencing library preparation protocol for low-input DNA samples can be used for low prevalence gut pathogens (< 0.1% of the total microbiome). This FACS-seq approach reduces diagnostics time (no culture is needed) and may promote discoveries of novel strains. Methodological details, possible issues and future directions for the sequencing of these natural pan-genomes are herein discussed.

Gut microbiota composition in Himalayan and Andean populations and its relationship with diet, lifestyle and adaptation to the high-altitude environment.

Human populations living at high altitude evolved a number of biological adjustments to cope with a challenging environment characterised especially by reduced oxygen availability and limited nutritional resources. This condition may also affect their gut microbiota composition. Here, we explored the impact of exposure to such selective pressures on human gut microbiota by considering different ethnic groups living at variable degrees of altitude: the high-altitude Sherpa and low-altitude Tamang populations from Nepal, the high-altitude Aymara population from Bolivia, as well as a low-altitude cohort of European ancestry, used as control. We thus observed microbial profiles common to the Sherpa and Aymara, but absent in the low-altitude cohorts, which may contribute to the achievement of adaptation to high-altitude lifestyle and nutritional conditions. The collected evidences suggest that microbial signatures associated to these rural populations may enhance metabolic functions able to supply essential compounds useful for the host to cope with high altitude-related physiological changes and energy demand. Therefore, these results add another valuable piece of the puzzle to the understanding of the beneficial effects of symbiosis between microbes and their human host even from an evolutionary perspective.

Draft Genome Sequences of 12 Monophasic Salmonella enterica subsp. enterica Serotype Typhimurium 1,4,[5],12:i:- Strains Isolated from Wild Griffon Vultures in Eastern Spain.

Monophasic Salmonella enterica subsp. enterica serovar Typhimurium is one of the most common zoonotic pathogens. Salmonella species reside in a wide variety of hosts, including wild animals. Thus, we report here the genome sequences of 12 monophasic S. Typhimurium strains isolated from healthy wild vultures to gain better insight into their epidemiology and host-pathogen interactions.

Mastiha (Pistacia lentiscus) Improves Gut Microbiota Diversity, Hepatic Steatosis, and Disease Activity in a Biopsy-Confirmed Mouse Model of Advanced Non-Alcoholic Steatohepatitis and Fibrosis.

SCOPE: As a result of the obesity epidemic, the prevalence of non-alcoholic steatohepatitis (NASH) is increasing. No drug is approved for the treatment of NASH. In this study, the effect of a nutritional supplement, Mastiha or Chios mastic gum, on metabolic and histological parameters and on the gut microbiome in mice with NASH and fibrosis was investigated. METHODS AND RESULTS: Advanced NASH was induced by feeding C57BL/6J mice a diet rich in fat, sucrose, and cholesterol for 41 weeks. After randomization, animals received the NASH-inducing diet with or without 0.2% (w/w) Mastiha for a further 8 weeks. Disease activity was assessed by liver histology and determination of plasma transaminase activities. Fecal microbiota DNA extraction and 16S rRNA amplicon sequencing were used to determine the composition of the gut microbiome. Mastiha supplementation led to a significant reduction in circulating alanine aminotransferase (ALT) activity, improvement in hepatic steatosis and collagen content, and a reduction in NAFLD activity score. Furthermore, it resulted in a partial but significant recovery of gut microbiota diversity and changes in identity and abundance of specific taxa. CONCLUSION: This is the first study demonstrating an improvement in disease activity in mice with advanced NASH with fibrosis by a diet containing Mastiha.

Natural history, dynamics, and ecology of human papillomaviruses in genital infections of young women: protocol of the PAPCLEAR cohort study.

INTRODUCTION: Human papillomaviruses (HPVs) are responsible for one-third of all cancers caused by infections. Most HPV studies focus on chronic infections and cancers, and we know little about the early stages of the infection. Our main objective is to better understand the course and natural history of cervical HPV infections in healthy, unvaccinated and vaccinated, young women, by characterising the dynamics of various infection-related populations (virus, epithelial cells, vaginal microbiota and immune effectors). Another objective is to analyse HPV diversity within hosts, and in the study population, in relation to co-factors (lifestyle characteristics, vaccination status, vaginal microbiota, human genetics). METHODS AND ANALYSIS: The PAPCLEAR study is a single center longitudinal study following 150 women, aged 18-25 years, for up to 2 years. Visits occur every 2 or 4 months (depending on HPV status) during which several variables are measured, such as behaviours (via questionnaires), vaginal pH, HPV presence and viral load (via qPCR), local concentrations of cytokines (via MesoScale Discovery technology) and immune cells (via flow cytometry). Additional analyses are outsourced, such as titration of circulating anti-HPV antibodies, vaginal microbiota sequencing (16S and ITS1 loci) and human genotyping. To increase the statistical power of the epidemiological arm of the study, an additional 150 women are screened cross-sectionally. Finally, to maximise the resolution of the time series, participants are asked to perform weekly self-samples at home. Statistical analyses will involve classical tools in epidemiology, genomics and virus kinetics, and will be performed or coordinated by the Centre National de la Recherche Scientifique (CNRS) in Montpellier. ETHICS AND DISSEMINATION: This study has been approved by the Comité de Protection des Personnes Sud Méditerranée I (reference number 2016-A00712-49); by the Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé (reference number 16.504); by the Commission Nationale Informatique et Libertés (reference number MMS/ABD/AR1612278, decision number DR-2016-488) and by the Agence Nationale de Sécurité du Médicament et des Produits de Santé (reference 20160072000007). Results will be published in preprint servers, peer-reviewed journals and disseminated through conferences. TRIAL REGISTRATION NUMBER: NCT02946346; Pre-results.

Spent Coffee Grounds Extract, Rich in Mannooligosaccharides, Promotes a Healthier Gut Microbial Community in a Dose-Dependent Manner.

Coffee is one of the most consumed beverages around the world, and as a consequence, spent coffee grounds are a massively produced residue that is causing environmental problems. Reusing them is a major focus of interest presently. We extracted mannooligosaccharides (MOS) from spent coffee grounds and submitted them to an in vitro fermentation with human feces. Results obtained suggest that MOS are able to exert a prebiotic effect on gut microbiota by stimulating the growth of some beneficial genera, such as Barnesiella, Odoribacter, Coprococcus, Butyricicoccus, Intestinimonas, Pseudoflavonifractor, and Veillonella. Moreover, short-chain fatty acids (SCFA) production also increased in a dose-dependent manner. However, we observed that 5-(hydroxymethyl)furfural, furfural, and polyphenols (which are either produced or released from the spent coffee grounds matrix during hydrolysis) could have an inhibitory effect on other beneficial genera, such as Faecalibacterium, Ruminococcus, Blautia, Butyricimonas, Dialister, Collinsella, and Anaerostipes, which could negatively affect the prebiotic activity of MOS.

Microbiota of sliced cooked ham packaged in modified atmosphere throughout the shelf life: Microbiota of sliced cooked ham in MAP.

Fourteen lots of cooked ham in modified atmosphere packaging (CH) were analyzed within a few days from packaging (S) and at the end of the shelf-life (E), after storage at 7 °C to simulate thermal abuse. Five more lots, rejected from the market because spoiled (R), were included in the study. Quality of the products was generally compromised during the shelf life, with only 4 lots remaining unaltered. Analysis of 16S rRNA gene amplicons resulted in 801 OTUs. S samples presented a higher diversity than E and R ones. At the beginning of the shelf life, Proteobacteria and Firmicutes dominated the microbiota, with Acinetobacter, Brochothrix, Carnobacterium, Lactobacillus, Prevotella, Pseudomonas, Psychrobacter, Weissella, Vibrio rumoiensis occurring frequently and/or abundantly. E and R samples were dominated by Firmicutes mostly ascribed to Lactobacillales. It is noteworthy the appearance of abundant Leuconostoc, negligible in S samples, in some E and R samples, while in other LAB were outnumbered by V. rumoiensis or Brochothrix thermosphacta. The microbiota of spoiled and R samples could not be clustered on the basis of specific defects (discoloration, presence of slime, sourness, and swollen packages) or supplemented additives. LAB population of S samples, averaging 2.9 log10(cfu/g), increased to 7.7 log10(cfu/g) in the E and R samples. Dominant cultivable LAB belonged to the species Lactobacillus sakei and Leuconostoc carnosum. The same biotypes ascribed to different species where often found in the corresponding S and R samples, and sometime in different batches provided from the same producer, suggesting a recurrent contamination from the plant of production. Consistently with growth of LAB, initial pH (6.26) dropped to 5.74 in E samples. Volatiles organic compound (VOCs) analysis revealed that ethanol was the major metabolite produced during the shelf life. The profile of volatile compounds got enriched with other molecules (e.g. 2-butanone, ethyl acetate, acetic acid, acetoin, butanoic acid, ethyl ester, butanoic acid, and 2,3-butanediol) mainly ascribed to microbial metabolism.

Oxidative stress in the oral cavity is driven by individual-specific bacterial communities.

The term "bacterial dysbiosis" is being used quite extensively in metagenomic studies, however, the identification of harmful bacteria often fails due to large overlap between the bacterial species found in healthy volunteers and patients. We hypothesized that the pathogenic oral bacteria are individual-specific and they correlate with oxidative stress markers in saliva which reflect the inflammatory processes in the oral cavity. Temporally direct and lagged correlations between the markers and bacterial taxa were computed individually for 26 volunteers who provided saliva samples during one month (21.2 ± 2.7 samples/volunteer, 551 samples in total). The volunteers' microbiomes differed significantly by their composition and also by their degree of microbiome temporal variability and oxidative stress markers fluctuation. The results showed that each of the marker-taxa pairs can have negative correlations in some volunteers while positive in others. Streptococcus mutans, which used to be associated with caries before the metagenomics era, had the most prominent correlations with the oxidative stress markers, however, these correlations were not confirmed in all volunteers. The importance of longitudinal samples collections in correlation studies was underlined by simulation of single sample collections in 1000 different combinations which produced contradictory results. In conclusion, the distinct intra-individual correlation patterns suggest that different bacterial consortia might be involved in the oxidative stress induction in each human subject. In the future, decreasing cost of DNA sequencing will allow to analyze multiple samples from each patient, which might help to explore potential diagnostic applications and understand pathogenesis of microbiome-associated oral diseases.