Low-Diversity Microbiota in Apical Periodontitis and High Blood Pressure Are Signatures of the Severity of Apical Lesions in Humans.

(1) Background: In developed countries, the prevalence of apical periodontitis (AP) varies from 20% to 50% for reasons that could be associated with the apical periodontitis microbiota ecology. (2) Methods: We performed a clinical study in the Odontology department of Toulouse hospital in France, to sequence the 16S rRNA gene of AP microbiota and collect clinical parameters from 94 patients. Forty-four patients were characterized with a PAI (periapical index of AP severity) score lower or equal to 3, while the others had superior scores (n = 50). (3) Results: The low diversity of granuloma microbiota is associated with the highest severity (PAI = 5) of periapical lesions (Odds Ratio 4.592, IC 95% [1.6329; 14.0728]; p = 0.001; notably, a lower relative abundance of Burkholderiaceae and a higher relative abundance of Pseudomonas and Prevotella). We also identified that high blood pressure (HBP) is associated with the increase in PAI scores. (4) Conclusions: Our data show that a low diversity of bacterial ecology of the AP is associated with severe PAI scores, suggesting a causal mechanism. Furthermore, a second risk factor was blood pressure associated with the severity of apical periodontitis.

Geographical and Seasonal Analysis of the Honeybee Microbiome.

We previously showed that colonies of thriving and non-thriving honeybees co-located in a single geographically isolated apiary harboured strikingly different microbiomes when sampled at a single time point in the honey season. Here, we profiled the microbiome in returning forager bees from 10 to 12 hives in each of 6 apiaries across the southern half of Ireland, at early, middle, and late time points in the 2019 honey production season. Despite the wide range of geographical locations and forage available, apiary site was not the strongest determinant of the honeybee microbiome. However, there was clear clustering of the honeybee microbiome by time point across all apiaries, independent of which apiary was sampled. The clustering of microbiome by time was weaker although still significant in three of the apiaries, which may be connected to their geographic location and other external factors. The potential forage effect was strongest at the second timepoint (June-July) when the apiaries also displayed greatest difference in microbiome diversity. We identified bacteria in the forager bee microbiome that correlated with hive health as measured by counts of larvae, bees, and honey production. These findings support the hypothesis that the global honeybee microbiome and its constituent species support thriving hives.

A synthetic consortium of 100 gut commensals modulates the composition and function in a colon model of the microbiome of elderly subjects.

Administration of cultured gut isolates holds promise for modulating the altered composition and function of the microbiota in older subjects, and for promoting their health. From among 692 initial isolates, we selected 100 gut commensal strains (MCC100) based on emulating the gut microbiota of healthy subjects, and retaining strain diversity within selected species. MCC100 susceptibility to seven antibiotics was determined, and their genomes were screened for virulence factor, antimicrobial resistance and bacteriocin genes. Supplementation of healthy and frail elderly microbiota types with the MCC100 in an in vitro colon model increased alpha-diversity, raised relative abundance of taxa including Blautia luti, Bacteroides fragilis, and Sutterella wadsworthensis; and introduced taxa such as Bifidobacterium spp. Microbiota changes correlated with higher levels of branched chain amino acids, which are health-associated in elderly. The study establishes that the MCC100 consortium can modulate older subjects' microbiota composition and associated metabolome in vitro, paving the way for pre-clinical and human trials.

Retention of Microbiota Diversity by Lactose-Free Milk in a Mouse Model of Elderly Gut Microbiota.

Prebiotics may improve aging-related dysbiosis. Milk is a source of nutrients including oligosaccharides whose prebiotic potential remains largely unexplored. We used a murine model to explore the effect of milk products on high diversity and lower diversity faecal microbiota from healthy and frail elderly subjects, respectively. Mice were treated with antibiotics and subsequently "humanized" with human faecal microbiota. The mice received lactose-free or whole milk, glycomacropeptide, or soy protein (control) supplemented diets for one month. The faecal microbiota was analyzed by 16S rRNA gene amplicon sequencing. Lactose-free milk diet was as efficient as the control diet in retaining faecal microbiota diversity in mice. Both milk diets had a significant effect on the relative abundance of health-relevant taxa (e.g., Ruminococcaceae, Lachnospiraceae). The glycomacropeptide prebiotic activity previously observed in vitro was not replicated in vivo. However, these data indicate the novel prebiotic potential of bovine milk for human nutrition.

Gut and Whole-Body Microbiota of the Honey Bee Separate Thriving and Non-thriving Hives.

The recent worldwide decline of honey bee colonies is a major ecological problem which also threatens pollinated crop production. Several interacting stressors such as environmental pressures and pathogens are suspected. Recently, the gut microbiota has emerged as a critical factor affecting bee health and fitness. We profiled the bacterial communities associated with the gut and whole body of worker bees to assess whether non-thriving colonies could be separated from thriving hives based on their microbial signature. The microbiota of thriving colonies was characterised by higher diversity and higher relative abundance of bacterial taxa involved in sugar degradation that were previously associated with healthy bees (e.g. Commensalibacter sp. and Bartonella apis). In contrast, the microbiota of non-thriving bees was depleted in health-associated species (e.g. Lactobacillus apis), and bacterial taxa associated with disease states (e.g. Gilliamella apicola) and pollen degradation (e.g. G. apicola and Bifidobacterium asteroides) were present in higher abundance compared to thriving colonies. Gut and whole-body microbiota shared a similar dominant core but their comparison showed differences in composition and relative abundance. More differences in taxon relative abundance between gut and whole body were observed in non-thriving bees, suggesting that microbiota associated with other bee organs might also be different. Thus, microbiota profiling could be used as a diagnostic tool in beekeeping practices to predict hive health and guide hive management.

Comparison of three methods for cell surface proteome extraction of Listeria monocytogenes biofilms.

The cell surface proteome of the foodborne pathogen Listeria monocytogenes, the etiological agent of listeriosis, is critical for understanding the physiological processes associated with stress resistance and persistence in the environment. In this context, the most widespread mode of growth for bacterial cells in natural and industrial environments is in biofilms. Cell surface proteins are, however, challenging to characterize because of their low abundance and poor solubility. Moreover, cell surface protein extracts are usually contaminated with cytoplasmic proteins that constitute the main signal in proteomic analysis. This study aimed to compare the efficiency of three methods to extract and explore surface proteins of L. monocytogenes growing in a biofilm: trypsin shaving, biotinylation, and cell fractionation. Peptide separation and identification were performed by shotgun proteomics using high-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). The biotinylation method was the most effective in extracting surface proteins, with the lowest rate of contamination by cytoplasmic proteins. Although presenting a higher contamination rate in cytoplasmic proteins, the other two techniques allowed the identification of additional surface proteins. Seven proteins were commonly retrieved by the three methods. The extracted proteins belong to several functional classes, involved in virulence, transport, or metabolic pathways. Finally, the three extraction methods seemed complementary and their combined use improved the exploration of the bacterial surface proteome. These new findings collectively inform future discovery and translational proteomics for clinical, environmental health, and industrial applications.

Comparing 16S rDNA amplicon sequencing and hybridization capture for pea aphid microbiota diversity analysis.

OBJECTIVE: Targeted sequencing of 16S rDNA amplicons is routinely used for microbial community profiling but this method suffers several limitations such as bias affinity of universal primers and short read size. Gene capture by hybridization represents a promising alternative. Here we used a metagenomic extract from the pea aphid Acyrthosiphon pisum to compare the performances of two widely used PCR primer pairs with DNA capture, based on solution hybrid selection. RESULTS: All methods produced an exhaustive description of the 8 bacterial taxa known to be present in this sample. In addition, the methods yielded similar quantitative results, with the number of reads strongly correlating with quantitative PCR controls. Both methods can thus be considered as qualitatively and quantitatively robust on such a sample with low microbial complexity.

Archaea: Microbial Candidates in Next-generation Probiotics Development.

Pharmabiotics and probiotics in current use or under development belong to 2 of 3 domains of life, Eukarya (eg, yeasts) and Bacteria (eg, lactobacilli). Archaea constitute a third domain of life, and are currently not used as probiotics, despite several interesting features. This includes the absence of known pathogens in humans, animals, or plants and the existence of some archaea closely associated to humans in various microbiomes. We promote the concept that some specific archaea that naturally thrive in the human gut are potential next-generation probiotics that can be rationally selected on the basis of their metabolic phenotype not being encountered in other human gut microbes, neither Bacteria nor Eukarya. The example of the possible bioremediation of the proatherogenic compound trimethylamine into methane by archaeal microbes is described.

Oral exposure to environmental pollutant benzo[a]pyrene impacts the intestinal epithelium and induces gut microbial shifts in murine model.

Gut microbiota dysbiosis are associated with a wide range of human diseases, including inflammatory bowel diseases. The physiopathology of these diseases has multifactorial aetiology in which environmental factors, particularly pollution could play a crucial role. Among the different pollutants listed, Polycyclic Aromatic Hydrocarbons (PAHs) are subject to increased monitoring due to their wide distribution and high toxicity on Humans. Here, we used 16S rRNA gene sequencing to investigate the impact of benzo[a]pyrene (BaP, most toxic PAH) oral exposure on the faecal and intestinal mucosa-associated bacteria in C57BL/6 mice. Intestinal inflammation was also evaluated by histological observations. BaP oral exposure significantly altered the composition and the abundance of the gut microbiota and led to moderate inflammation in ileal and colonic mucosa. More severe lesions were observed in ileal segment. Shifts in gut microbiota associated with moderate inflammatory signs in intestinal mucosa would suggest the establishment of a pro-inflammatory intestinal environment following BaP oral exposure. Therefore, under conditions of genetic susceptibility and in association with other environmental factors, exposure to this pollutant could trigger and/or accelerate the development of inflammatory pathologies.

Targeted Gene Capture by Hybridization to Illuminate Ecosystem Functioning.

Microbial communities are extremely abundant and diverse on earth surface and play key role in the ecosystem functioning. Thus, although next-generation sequencing (NGS) technologies have greatly improved knowledge on microbial diversity, it is necessary to reduce the biological complexity to better understand the microorganism functions. To achieve this goal, we describe a promising approach, based on the solution hybrid selection (SHS) method for the selective enrichment in a target-specific biomarker from metagenomic and metatranscriptomic samples. The success of this method strongly depends on the determination of sensitive, specific, and explorative probes to assess the complete targeted gene repertoire. Indeed, in this method, RNA probes were used to capture large DNA or RNA fragments harboring biomarkers of interest that potentially allow to link structure and function of communities of interest.

The Prediction and Validation of Small CDSs Expand the Gene Repertoire of the Smallest Known Eukaryotic Genomes.

The proper prediction of the gene catalogue of an organism is essential to obtain a representative snapshot of its overall lifestyle, especially when it is not amenable to culturing. Microsporidia are obligate intracellular, sometimes hard to culture, eukaryotic parasites known to infect members of every animal phylum. To date, sequencing and annotation of microsporidian genomes have revealed a poor gene complement with highly reduced gene sizes. In the present paper, we investigated whether such gene sizes may have induced biases for the methodologies used for genome annotation, with an emphasis on small coding sequence (CDS) gene prediction. Using better delineated intergenic regions from four Encephalitozoon genomes, we predicted de novo new small CDSs with sizes ranging from 78 to 255 bp (median 168) and corroborated these predictions by RACE-PCR experiments in Encephalitozoon cuniculi. Most of the newly found genes are present in other distantly related microsporidian species, suggesting their biological relevance. The present study provides a better framework for annotating microsporidian genomes and to train and evaluate new computational methods dedicated at detecting ultra-small genes in various organisms.

Capturing prokaryotic dark matter genomes.

Prokaryotes are the most diverse and abundant cellular life forms on Earth. Most of them, identified by indirect molecular approaches, belong to microbial dark matter. The advent of metagenomic and single-cell genomic approaches has highlighted the metabolic capabilities of numerous members of this dark matter through genome reconstruction. Thus, linking functions back to the species has revolutionized our understanding of how ecosystem function is sustained by the microbial world. This review will present discoveries acquired through the illumination of prokaryotic dark matter genomes by these innovative approaches.

Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment.

PhylOPDb: a 16S rRNA oligonucleotide probe database for prokaryotic identification.

In recent years, high-throughput molecular tools have led to an exponential growth of available 16S rRNA gene sequences. Incorporating such data, molecular tools based on target-probe hybridization were developed to monitor microbial communities within complex environments. Unfortunately, only a few 16S rRNA gene-targeted probe collections were described. Here, we present PhylOPDb, an online resource for a comprehensive phylogenetic oligonucleotide probe database. PhylOPDb provides a convivial and easy-to-use web interface to browse both regular and explorative 16S rRNA-targeted probes. Such probes set or subset could be used to globally monitor known and unknown prokaryotic communities through various techniques including DNA microarrays, polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), targeted gene capture or in silico rapid sequence identification. PhylOPDb contains 74 003 25-mer probes targeting 2178 genera including Bacteria and Archaea. Database URL: http://g2im.u-clermont1.fr/phylopdb/